Gene expression studies in prostate cancer tissue: which reference gene should be selected for normalization?

Abstract

Using quantitative reverse transcription-polymerase chain reaction (RT-PCR), reference genes are utilized as endogenous controls for relative quantification of target genes in gene profiling studies. The suitability of housekeeping genes for that purpose in prostate cancer tissue has not been sufficiently investigated so far. The objective of this study was to select from a panel of 16 potential candidate reference genes the most stable genes for gene normalization. Expression of mRNA encoding ACTB, ALAS1, ALB, B2M, G6PD, GAPD, HMBS, HPRT1, K-ALPHA-1, POLR2A, PPIA, RPL13A, SDHA, TBP, UBC, and YWHAZ was examined in matched, microdissected malignant and nonmalignant tissue specimens obtained from 17 nontreated prostate carcinomas after radical prostatectomy by real-time RT-PCR. The genes studied displayed a wide expression range with cycle threshold values between 16 and 37. The expression was not different between samples from pT2 and pT3 tumors or between samples with Gleason scores <7 and >or=7 (P>0.05). ACTB, RPL13A, and HMBS showed significant differences (P<0.02 at least) in expressions between malignant and nonmalignant pairs. All other genes did not differ between the matched pairs, and the software programs geNorm and NormFinder were used to ascertain the most suitable reference genes from these candidates. HPRT1, ALAS1, and K-ALPHA-1 were calculated by both programs to be the most stable genes covering a broad range of expression. The expression of the target gene RECK normalized with HRPT1 alone and with the normalization factors generated by the combination of these three reference genes as well as with the unstable genes ACTB or RPL13A is given. That example shows the significance of using suitable reference genes to avoid erroneous normalizations in gene profiling studies for prostate cancer. The use of HPRT1 alone as a reference gene shown in our study was sufficient, but the normalization factors generated from two (HRPT1, ALAS1) or all three genes (HRPT1, ALAS1, K-ALPHA-1) should be considered for an improved reliability of normalization in gene profiling studies of prostate cancer.

The Fer Tyrosine Kinase Cooperates with Interleukin-6 to Activate Signal Transducer and Activator of Transcription 3 and Promote Human Prostate Cancer Cell Growth

“…For cDNA synthesis, 1 Ag RNA was reverse transcribed using Expand Reverse transcriptase (Roche), according to the manufacturer's protocol. Samples (5 AL of cDNA in a 50 AL final volume) were used to quantify levels of fer transcripts by real-time PCR relative to levels of succinate dehydrogenase (SDHA) used as a reference housekeeping gene (53). PCRs were carried out using validated primers for Fer (QT00029043) and SDHA (QT00059486) with the QuantiTect SYBR Green PCR Master Mix, according to the manufacturer's instructions (Qiagen), in a MyiQ Single-Color Real-Time PCR Detection System (Bio-Rad).…”

Evaluation and validation of suitable reference genes for reverse transcription-quantitative polymerase chain reaction studies in cholangiocarcinoma patients and cell lines

“…grade and stage of tumors, and as previous studies indicate, the expression of a reference gene is not directly associated with the grade and stage of malignant tumors (21); the selected samples were all characterized as malignant tissue samples by the pathology department of the China-Japan Union Hospital of Jilin University, and the pathology observed in the samples indicated the most common pathological type of CCA, adenocarcinoma; the QBC939 cell line was also used as it is a commonly used human CCA cell line; the stability evaluation on reference genes for the selected histological samples and cell lines of human CCA was implemented to make the experimental results more comprehensive, compared with previous studies (9,10,21,22) where the stability evaluation on reference genes was restricted on ceratin tissues or cell lines; and the stability of 12 common reference genes was compared.…”

“…Therefore, during the detection of target gene expression, a gene with a stable expression is required as a reference. Recent studies have revealed defects in all commonly used reference genes, which often vary greatly in expression quantity in various types of cells and tissues, stages of cell amplification and organ development and in vitro culture and experimental conditions (9,10,(20)(21)(22). To the best of our knowledge, there have been no systematic comparisons performed concerning the stability of commonly-used reference genes in CCA tissues and cell lines.…”

“…However, the analysis results of BestKeeper are different to those observed with GeNorm and NormFinder. Previous studies hypothesize that this may be due to differences in the statistical model that BestKeeper uses; therefore, it is less effective for ranking reference gene stability compared with GeNorm and NormFinder (20)(21)(22). Based on comparisons of results among the three programs, the present study recommends the following most stable reference genes, which have been confirmed through analysis by ≥2 programs: Total samples group, RPL29; CCA tissue group, TBP; CCA cell line and CCA tissue group, ACTB; CCA cell line group, 18SrRNA; CCA tissue and adjacent non-neoplastic tissue group, ALAS1; and adjacent non-neoplastic tissue group, HPRT1.…”

Zebrafish GAPDH can be used as a reference gene for expression analysis in cross-subfamily cloned embryos

“…A reliable reference gene for the transcriptional study in cross-subfamily cloned embryo is necessary for comparative analysis. Previous studies had provided solid evidence that the expression levels of some housekeeping genes varied considerably in diVerent developmental stages and under diVerent experimental conditions [3][4][5]. In the present study, we analyzed GAPDH (glyceraldehyde-3-phosphate dehydrogenase) expression levels both in zebraWsh embryos and in cross-subfamily cloned embryos to determine whether GAPDH could be used as a suitable reference for nuclear reprogramming.…”